All of the folded components of polyketide synthase (PKS) modules have now been structurally characterized, yet the quintessential three-dimensional puzzle of the multimodular PKS assembly line (d8 MDa) has still not been solved. Our limited understanding of how synthase components structurally and enzymatically interface with one another is the major gap in our knowledge. In order to realize our long-term goal of accelerating the development of natural products into new antibiotics and anticancer agents by engineering multimodular PKSs to synthesize combinatorial libraries of promising polyketide drug leads this information must be elucidated. We are in the home stretch in determining the architectures and activities of these largest known enzymes, thus our current goal is to solve the multimodular PKS assembly line puzzle through determining each of its domain- domain interfaces (i.e. how the individual pieces fit together). From the atomic-resolution structures tha have been reported as well as several architecturally-informative structures not yet reported from our lab, we have constructed models of modules and bimodules that are consistent with all the available biochemical, biophysical, and bioinformatics data. While for many years scientists have sought the crystal structure of a PKS module, our models suggest that each module has a flexible waist region like that of the related mammalian fatty acid synthase and that the major interactions within PKS assembly lines are actually across modular boundaries. Thus, we will be guided by our models towards obtaining the physical data of how domains assemble and test our central hypothesis that the structures of PKS components are altered and enzymatic activities are enhanced through domain interactions formed within an intact synthase. We first seek to observe the most relevant multidomain complexes through x-ray crystallography (Specific Aim 1). Other biophysical techniques such as small-angle x-ray scattering (SAXS) and sedimentation velocity analytical ultracentrifugation that do not rely on crystal formation are als very powerful tools, especially now that the atomic-resolution structures of each synthase component have been determined. Thus, even if crystals of desired complexes are not obtained, domain interfaces will be identified through a combination of biophysical techniques and site-directed mutagenesis (Specific Aim 2). We will also functionally probe the architecture of PKS modules through an innovative approach developed in my lab that utilizes biocatalytic and chemical biology tools to fluorescently label products of PKS modules. How component enzymes respond to mutations at suspected interfaces and the shortening of key flexible linkers will help reveal many desired structural and functional details of PKS modules (Specific Aim 3). Our proposed research is significant as multimodular PKSs produce many important human medicines, such as the antibacterial erythromycin, the antifungal amphotericin, and the anticancer agent epothilone, and through an increased understanding of how these molecular factories operate we will be able to utilize them in the more rapid development of new antibiotics and anticancer drugs.